Abstract/Project Summary The placenta is of paramount importance for reproductive success and placental defects present a major reproductive health challenge. The overall objective of the proposed work is to decode the regulatory circuitry of trophoblast differentiation that ensures proper placental development in humans and mice, a crucial but poorly understood area. BCOR encodes a component of a PRC1 Polycomb group repression complex that is essential for placental development in the mouse and it provides a powerful tool to identify new key developmental regulators of the circuitry underlying this process. The central hypothesis of this proposal is that the BCOR-containing PRC1 complex is differentially deployed in trophoblast stem/progenitor cells and their progeny to silence key lineage-determining genes of alternate cell fates. This cell type-specific targeting of BCOR ensures that the trophoblastic cell types are established and maintain their differentiated fates and are present in appropriate numbers. This proposal will be able to get to the heart of early BCOR complex function from both developmental and mechanistic standpoints in both mouse and human due to the availability of many key in vivo and in vitro systems and reagents, including a Bcor conditional mouse allele and stem cell lines allowing conditional deletion of Bcor. The studies proposed here will tackle three key questions in three aims whose answers will fill major knowledge gaps about the circuitry of trophoblast differentiation 1) How does Bcor regulate the allocation of progenitor and differentiated cell fates in vivo? This aim will determine the fundamental in vivo role of Bcor in placental development and cell fate specification with a focus on progenitor cell compartments and cell type allocation. 2) Which networks of lineage-specific BCOR targets underlie the trophoblast differentiation circuitry and explain the Bcor mutant placental phenotype? This aim will define the BCOR functional targets in mouse cultured TS cells, primary human trophoblasts and BMP4-treated hESCs, and their differentiated progeny. Bioinformatic analysis of these data will identify key regulators of trophoblast differentiation and help define the regulatory architecture underlying placental lineage specification and how BCOR controls it. 3) What are the lineage-specific transcription factors (TF) that direct cell type-specific targeting of the BCOR complex? This aim will identify BCOR complex-associated transcription factors in mouse and human trophoblast cell types and then test whether they contribute to cell type-specific recruitment to target genes. Success in these aims will generate fundamental knowledge about the circuitry underlying trophoblast differentiation and lineage-specific recruitment mechanisms of PcG complexes. This basic information will lead not only to a deeper understanding of early placental development, a process vital for health and reproduction, but also promises to inform the design of approaches to control the differentiation of stem cells.